​​​​​​​​​​​​March 2018, Vol. 3, No. 3, pp 65-71. 

​​Heterologous Expression of Exoglucanase from Trichoderma resei in E. coli

R. Navnit kumar¹, S. Jason Charles², T. R. Sambavi¹, S. Kabilan³, S. Renganathan¹,*
¹Biofuels Laboratory, Centre for Biotechnology, Anna University, Chennai. India.
²Molecular Biology Laboratory, Centre for Biotechnology, Anna University, Chennai. India.
³Bioprocess Laboratory, Centre for Biotechnology, Anna University, Chennai. India.
​​​*Corresponding author’s e-mail: rengsah@rediffmail.com

​Abstract

​FPases/Exoglucanases are usually produced in lesser quantity from the wild cellulolytic strains in comparison to the quantities of Endoglucanases, Beta glucosidases and Xylanases. In the present work, an intracellular soluble expression of exoglucanase was attempted in a prokaryotic expression host E. coli SHuffle. A gene Cel7a that codes for Exgolucanases/FPases was isolated from Trichoderma resei and ligated into the pRSET B vector. E. coli SHuffle strain was transformed with the plasmid vector. A His-Tag metal affinity purification was performed after a fermentation that lasted for 3.5 hr using the recombinant E.coli Shuffle strain. Around 0.7 g/L of exoglucanases of 58 KDa molecular weight was obtained after the purification. The recombinant Exoglucanase had an activity of around 2.5 FPU/mL when assessed using the standard IUPAC Ghose assay for cellulase.

Keywords: Bioethanol; Fermentation; Cellulase; Vector; Prokaryotic expression.

​References

  1. ​Parisutham V, Lee SK. Heterologous Expression and Extracellular Secretion of Cellulases in Recombinant Microbes. Marco Aurelio PL, Alexandra Pardo PN (eds.) Bioehanol. InTech Open. 2012.
  2. Salwee Y, Mattoo RL, Nehvi FA. Isolation, Characterization and Molecular weight determination of Cellulase from Trichoderma viride. African Journal of Biotechnology. 2013;12:4512-4518.
  3. Camilla L, Megan G, Johannes K, Dirk H, Holger K, Rainer F, Ulrich C. Challenges and Advances in the Heterologous Expression of Cellulolytic Enzymes: A Review. Biotechnology for Biofuels. 2014. 7:135. https://doi.org/10.1186/s13068-014-0135-5
  4. Andrew SW, Damien BP, Vincent JJM. Recombinant Cellulase and Cellulosome Systems. Cellulose-Biomass Conversion. Prof. John Kadla (Ed.), InTech Open. 2013.
  5. Rodrigues AL, Cavalett A, Lima AOS. Enhancement of Escherichia coli cellulolytic activity by co-production of beta-glucosidase and endoglucanase enzymes. Electronic Journal of Biotechnology. 2010;13(5): DOI: 10.2225/vol13-issue5-fulltext-19.
  6. Sibtain A, Ammara B, Huma S, Mubshara S, Amer J. Production and Purification of Cellulose Degrading Enzymes from a Filamentous Fungus Trichoderma harzianum. Pak J Bot. 2009;41:1411-1419.
  7. Sonika Pandey, Mukesh Srivastava, Mohammad Shahid, Vipul Kumar, Anuradha Singh, Shubha Trivedi and Y.K. Srivastava. Trichoderma species Cellulases Produced by Solid State Fermentation. Data Mining in Genomics and Proteomics. 2015. 6:2.
  8. Mina ER, Morteza A, Aftab B, Vladimir VB. A Novel Vector for Expression/Secretion of Properly Folded Eukaryotic Proteins: a Comparative Study on Cytoplasmic and Periplasmic Expression of Human Epidermal Growth Factor in E. coli. Iranian Biomedical Journal. 2004;8(2):51-61.
  9. Venkatesh. S and John Vennison. S. Cloning and expression of Exoglucanase gene from Trichoderma reesei in to Escherichia coli. International Journal of Current Biotechnology. 2016;4(12):1-6.
  10. Zehra Y, Eda Ç. Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells. Society of Chemical Industry. 2016;92(2):319-324.
  11. Amraini SZ, Ariyani LP, Hermansyah H, Setyahadi S, Rahman SF, Park DH, Gozan M. Production and Characterization of Cellulase from E. coli EgRK2 Recombinant Based on Oil Palm Empty Fruit Bunches. Biotechnology and Bioprocess Engineering. 2017;22:287-295.
  12. Varedi Koolaee SM, Shojaosadati SA, Babaeipour V, Ghaemi N. Physiological and morphological changes of recombinant E. coli during over-expression of human interferon-γ γ in HCDC. Iranian Journal of Biotechnology. 2006;4(4);230-238.
  13. pRSET A, B, and C For high-level expression of recombinant proteins in E. coli. Cat. No. V351-20. Invitrogen. Thermo Fisher Scientific: 2010.
  14. Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. Shuffle a Novel Escherechia coli protein Expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial Cell Factories. 2012;11:56. DOI: 10.1186/1475-2859-11-56.
  15. Yesubabu, Elavazhagan T. Expression of Eukaryotic Gene in E. coli. Middle East Journal of Scientific Research. 2009;4(3):133-136.   
  16. Sharma A, Chaudhuri TK. Revisiting Escherichia coli as microbial factory for enhanced production of human serum albumin. Microbial Cell Factories. 2017:16:173. https://doi.org/10.1186/s12934-017-0784-8
  17. Ghose TK. Measurement of Cellulase Activities. Pure App Chem. 1987;59(2):257-268.
  18. Bakri Y, Akeed Y, Thonart P. Comparison between Continuous and Batch Processing to Produce Xylanase by Penicillium canescens 10-10c. Brazilian Journal of Chemical Engineering. 2012;29(3): 441-448.
  19. Burger S, Tatge H, Hofmann F, Genth H, Just I, Gerhard R. Expression of recombinant Clostridium difficile toxin A using the Bacillus megateriu. Biochem Biophys Res Commun.  2012;307(3):584-588.
  20. Meenu K, Singh G, Vishwakarma RA. Molecular Mechanism of Cellulase Production Systems in Trichoderma. Gupta VK, Monika S, Alfredo HE, Upadhyay RS, Druzhinina I, Tuohy MG (eds.). Biotechnology and Biology of Trichoderma. Elsevier. 2014.

International Journal of Modern Science and Technology

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ISSN 2456-0235